Prolactin (“PRL”) is a 23-kDa neuroendocrine hormone which is structurally related to growth hormone and, to a lesser degree, to members of the interleukin family (Reynolds et al., 1997, Endocrinol. 138:5555–5560, Cunningham et al., 1990, Science 247:1461–1465; Wells et al., 1993, Recent Prog. Horm. Res. 48:253–275). Acting via the prolactin receptor, it is required for the proliferation and terminal differentiation of breast tissue (Mani et al., 1986, Cancer Res. 46:1669–1672; Malarkey et al., 1983, J. Clin. Endocrinol. Metab. 56:673–677; Biswas and Vonderhaar, 1987, Cancer Res. 47:3509–3514), promoting the growth and differentiation of the ductal epithelium, proliferation and differentiation of lobular units, and initiation and maintenance of lactation (Kelly et al., 1993, Recent Prog. Horm. Res. 48:123–164; Shiu et al., 1987, recent Pro. Horm. Res. 43:277–303). A diversity of other effects have been attributed to PRL, including roles in reproduction and the immune response (Wennbo et al., 1997, Endocrinol. 138:4410–4415; Nicoll, 1974, in Handbook of Physiology, Knobil and Sawyer, eds., American Physiological Society, Washington, D.C.; Shiu and Friesen, 1980, Annu. Rev. Physiol. 42:83–96).
The prolactin receptor (“PRLR”) is a member of the cytokine receptor superfamily and binds a group of hormones, including not only PRL but also placental lactogens and primate growth hormone (“GH”), to produce a mitogenic effect (Ormandy et al., 1997, J. Clin. Endocrinol. Metab. 82:3692–3699; Horseman, 1995, Endocrinol. 136:5249–5251; Clevenger et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:6460–6464; Buckley et al., 1985, Life Sci. 37:2569–2575; Costello et al., 1994, Prostate 24:162–166). PRLR is homologous to the receptor for GH (“GHR”, also referred to as the somatogen receptor) and both belong to the cytokine receptor superfamily (Kelly et al., 1991, Endocrin. Rev. 12:235–251; Kelly et al., 1993, Recent. Prog. Horm. Res. 48:123–164; Horseman and Yu-Lee, 1994, Endocrin. Rev. 15:627–649).
An association between PRL activity and breast cancer has been proposed (Ormandy et al., 1997, J. Clin. Endocrinol. Metab. 82:3692–3699). Elevated PRL levels have been found to accelerate the growth of mammary tumors induced by 7,12 dimethylbenz(α)antracene in rats, whereas PRL ablation was observed to have an inhibitory effect (Welsch, 1985, Cancer Res. 45:3415–3443). Mammary tumor growth was increased in transgenic mice overexpressing human GH, which binds to the rodent PRLR (Bartke et al., 1994, Proc. Soc. Exp. Biol. Med. 206:345–359). It has been found that the receptors for sex steroids and PRL are co-expressed and cross-regulated, which might explain the synergistic actions of estrogen, progesterone, and PRL in tumor growth control (Ormandy et al., 1997, J. Clin. Endocrinol. Metab. 82:3692–3699).
Nevertheless, to date, therapies which reduce PRL levels, such as hypophysectomy and bromocriptine administration (both directed toward decreasing or eliminating production of PRL by the pituitary gland), have not been successful in the treatment of breast cancer (Peyrat et al., 1984, Eur. J. Cancer Clin. Oncol. 20:1363–1367; Heuson et al., 1972, Eur. J. Cancer 8:155–156). It has been proposed that PRL may nevertheless have a role in breast cancer if an autocrine/paracrine growth regulatory loop exists (that is to say, that the pituitary is only one of several sources for prolactin; see Clevenger et al., 1995, Am. J. Pathol. 146:695–705, Fields et al., 1993, Lab. Invest. 68:354–360; Ginsburg and Vonderhaar, 1995, Cancer Res. 55:2591–2595; Fuh and Wells, 1995, J. Biol. Chem. 270:13133–13137). In this regard, when RNA levels of PRL and PRLR were performed using reverse transcriptase/PCR techniques, it was found that PRL and PRLR were widely expressed in breast cancers (>95 percent) and normal breast tissues (>93 percent), suggesting that interventions in the PRL/PRLR receptor may be useful in the treatment of breast cancer (Reynolds et al., Endocrinol. 138:5555–5560). Indeed, it has recently been reported that a combined regimen combining an anti-estrogen (tamoxifen), a GH analog (octreotide), and a potent anti-prolactin (CV 205-502, a dopamine agonist which inhibits prolactin secretion by the pituitary) had better clinical results in metastatic breast cancer patients compared to tamoxifen therapy alone (Botenbal et al., 1998, Br. J. Cancer 77:115–122).
An association between PRL expression and prostate disease has also been proposed (Wennbo et al., 1997, Endocrinol. 138:4410–4415). PRL receptors are found in prostate tissue (Aragona and Friesen, 1975, Endocrinol. 97:677–684; Leake et al., 1983, J. Endocrinol. 99:321–328). PRL levels have been observed to increase with age (Hammond et al., 1977, Clin. Endocrinol. 7:129–135; Vekemans and Robyn, 1975, Br. Med. J. 4:738–739) coincident with the development of prostate hyperplasia and PRL has been found to have trophic and differentiating effects on prostate tissue (Costello and Franklin, 1994, Prostate 24:162–166). Transgenic mice overexpressing the PRL gene developed dramatic enlargement of the prostate gland (Wennbo et al., 1997, Endocrinol. 138:4410–4415). Nonetheless, the role for PRL in prostate disease remains unclear (Wennbo et al., 1997, Endocrinol. 138:4410–4415). PRL levels in patients having prostate hyperplasia have been reported to be either increased (Odoma et al., 1985, J. Urol. 133:717–720; Saroff et al., 1980, Oncology 37:46–52), increased only in patients with prostate cancer or unchanged (Harper et al., 1976, Acta Endocrinol. (Copenh) 81:409–426). Janssen et al. reported that proliferation of androgen-insensitive human prostate cell lines can be significantly modulated by PRL (1996, Cancer 77:144–149). To explain these discrepancies, it has been proposed that local synthesis of PRL in the prostate (Nevalainen et al., 1997, J. Clin. Invest. 99:618–627) may be an important factor. Androgen-dependent expression of PRL in rat prostate epithelium has been observed, supporting the concept of an autocrine/paracrine loop of prolactin action in the prostate, where it could mediate androgen-associated effects (Nevalainen et al., 1997, FASEB J. 11(14):1297–1307). Further, clinical data appears promising: hypophysectomy has been found to have an additive therapeutic effect when combined with castration and adrenalectomy in prostate cancer patients (Brendler, 1973, Urology 2:99–102), and Rana et al. report that a combined maximal suppression of androgens and prolactin resulted in a significantly improved clinical response over conventional treatments in patients suffering from advanced prostate cancer (Habib et al., 1995, Eur. J. Cancer 31 A:859–860).
In view of the biological relevance of the PRL molecule and its receptor, a number of investigators have evaluated the activity of PRL variants which bear structural differences relative to the native unmodified molecule. It has been reported that naturally phosphorylated rat PRL antagonizes the growth-promoting effects of unmodified PRL in an assay which measures proliferation of rat Nb2 T lymphoma cells and in the autocrine regulation of GH3 cell proliferation (Wang and Walker, 1993, Endocrinol. 133:2156–2160; Krown et al., 1992, Endocrinol. 122:223–229). Further, molecular mimics of phosphorylated PRL having a bulky negatively charged amino acid (namely glutamate or aspartate) substituted for the serine at position 179 antagonized the growth-promoting effects of PRL (Chen et al., 1998, Endocrinol. 139: 609–616).
Other strategies for PRL variant design have been directed at disruption of the interaction between PRL and its receptor. To this end, researchers have drawn analogies between the PRLR and the GHR, for which the structure/function relationships are better understood.
Certain features of the GHR were elucidated by studying the basis for the full GH antagonist activity of the variant of human GH (“hGH”) having a substitution of the glycine at position 120 with an arginine residue (Chen et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:5061–5065; Chen et al., 1991, Mol. Endocrinol. 5:1845–1852; Chen et al., 1994, J. Biol. Chem. 269:15892–15897; Chen et al., 1995, Mol. Endocrinol. 9:1–7; U.S. Pat. No. 5,350,836 by Kopchick and Chen; U.S. Pat. No. 5,681,809 by Kopchick and Chen). It was deduced that hGH forms a complex with a dimeric form of the hGHR. Fuh and colleagues proposed a sequential dimerization model whereby GH would first bind to one receptor via a first binding site (delimited by portions of helix 1, helix 4 and loop 1 of GH) to form an inactive intermediate 1:1 complex, and then the receptor-bound hGH would interact with a second receptor through binding site 2 (involving the helix 3 glycine of GH mutated in the G120R variant) to produce the active 1:2 hormone/receptor complex (Fuh et al., 1992, Science 256:1677–1680; Fuh et al., 1993, J. Biol. Chem. 268:5376–5381, Goffin et al., 1994, J. Biol. Chem. 269:32598–32606). When the helix 3 glycine at position 120 of GH is substituted with an arginine residue, the second binding site is sterically hindered and the GH can no longer induce receptor dimerization.
Although less is known about the structure of the PRLR, it has been suggested that it, too, is activated by hormone-mediated sequential dimerization (Cunningham et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:3407–3411; Fuh et al., 1992, Science 256: 1677–1680; Fuh et al., 1993, J. Biol. Chem. 268:5376–5381). Variants of human PRL (“hPRL”) were produced containing mutations in the region believed to correspond to the helix 3/helix 1 interface of GH, including mutations of the alanine at position 22, the leucine at position 25, the serine at position 26 and the glycine at position 129 of PRL to tryptophan and/or arginine (specifically, to create A22W, L25R, L25W, S26R, S26W and G129R; Goffin et al., 1994, J. Biol. Chem. 269:32598–32606). It was reported in that paper that the point mutations at A22, S26 and G129 drastically decreased the mitogenic potency of the variant (as compared to native PRL) by 2–3 orders of magnitude (as tested in the Nb2 proliferation assay), although the G129R variant (positionally analogous to G120R of GH) was reported to act as a weak agonist rather than as an antagonist. It was subsequently reported that when tested in an assay for PRLR activity in which cells, co-transfected with nucleic acid encoding the hPRLk and a reporter gene under the control of PRL-responsive DNA sequences, were exposed to the G129R hPRL variant, an antagonist effect was observed (Goffin et al., 1996, J. Biol. Chem. 271:16573–16579).
Naturally occurring antagonists of GH action may exist. A cell-free truncated form of the GHR (termed “GH-BP”) has been identified in man and certain animals (Baumann, 1991, Acta Endocrinol. 124(suppl 2):21–26; Baumann et al., 1994, J. Endocrinol. 141:1–6; Baumann, 1995, Endocrinol. 136:377–378). The human form of GH-BP encompasses the extracellular domain of the receptor, and could be the result of proteolytic cleavage of the native receptor or alternative RNA splicing. It has been suggested that GH-BP acts to inhibit binding of GH to its receptors (Baumann, 1991, Acta Endocrinol. 124(suppl 2):21–26; Baumann et al., 1994, J. Endocrinol. 141:1–6). Supportive of this hypothesis is the observation that GH-BP levels in patients suffering from acromegaly (due to overexpression of GH) have an inverse correlation with serum GH levels (that is to say, the less GH-BP, the more serum GH present; Amit et al., 1992, Hormone Res. 37:205–211). Lower levels of GH-BP may render the acromegalic serum GH relatively more active in the GH receptor assay and therefore contribute negatively to the disease (Hochberg et al., 1994, Acta Endocrinol. 125:23–27). Soluble forms of other receptors in the cytokine receptor superfamily have also been observed (Baumann, 1995, Endocrinol. 136:377–378). Nevertheless, there has not been, prior to the present invention, any evidence suggesting the existence of a naturally occurring cell-free from of the PRLR.